Case 1 Single-cell sequencing to analyse differences in gene expression in different cells of the kidney
The relationship between gut microbial dysbiosis and acute or chronic kidney disease (CKD) is still unclear. This study showed that oral administration of the probiotic Lactobacillus casei Zhang (L. casei Zhang) corrected bilateral renal ischemia-reperfusion (I/R)-induced gut microbial dysbiosis, alleviated kidney injury, and delayed its progression to CKD in mice. L. casei Zhang elevated the levels of short-chain fatty acids (SCFAs) and nicotinamide in the serum and kidney, resulting in reduced renal inflammation and damage to renal tubular epithelial cells.
The authors used single-cell RNA sequencing (sScRNA-seq) to analyse differential gene expression in proximal tubules, macrophages, neutrophils and fibroblasts in the kidneys of I/R d5 and I/R d5+Lac.z mice and found that macrophages, neutrophils and fibroblasts in the I/R d5 group showed more pro-inflammatory and pro-fibrotic gene expression. In contrast, proximal tubule cells in the I/R d5+Lac.z group showed higher levels of expression of genes involved in repair compared to the I/R d5 group. Analysis of the distribution of SCFAs-related receptors and transporter proteins in the cell population revealed that the SCFAs-related transporter proteins SIC5a8 and SIC18a1 were mainly expressed in proximal tubule cells. The related receptors GPR43 and GPr109a were mainly expressed in fibroblasts, neutrophils and macrophages. These results suggest that oral administration of L. casei Zhang is a potential way to reduce renal injury and slow renal decline by altering SCFAs and nicotinamide metabolism.
Reference：Zhu H , Cao C , Wu Z , et al. The probiotic L. casei Zhang slows the progression of acute and chronic kidney disease (vol 33, 1.e1, 2021)[J]. Cell metabolism, 2021(10):33.
Case 2 Transcriptomic profiling of single extracellular vesicles by single cell RNA sequencing
A high-throughput sequencing method for vesicles (EVs) based on the 10x Genomics platform has been proposed by the An-Yuan Guo team at Huazhong University of Science and Technology (HUST), which is capable of probing the transcriptomic signature of EVs at the single EV level. The researchers used Calcein-AM to label intact EVs of K562 cell and mesenchymal stem cell, and detected the concentration of EVs samples by flow cytometry. The isolated single EVs were sequenced using the 10x Genomics platform. During data analysis, the team went through various trials and refinements and found that the CB2 algorithm using adaptive thresholding was effective in distinguishing true EVs from the background.
Reliable QC parameters were used to remove low quality data and DoubletFinder software was used to remove potential doublets. Ultimately, transcriptomic data were obtained for 2,088, 3,935 and 1,603 EVs in three different EV samples.
Analysis of the EV transcriptomes revealed that the average number of mRNA genes contained in a single EV was 52, ranging from 6 to 148 for different EVs. A higher proportion of EVs contained ribosomal genes, mitochondrial genes and EEF1A1 genes. K562 cell-derived EVs contained a high proportion of haemoglobin genes, while MSC-derived EVs were rich in cytoskeleton-related genes. Furthermore, through data downscaling and clustering analysis, the team demonstrated that EVs of even homogeneous cell origin are highly heterogeneous and can be divided into multiple subgroups of EVs with specific genes. This study revealed the transcriptome characteristics and heterogeneity of EVs at the single EV level by a high-throughput approach for the first time, refreshing the understanding of the RNA contents in individual EVs and providing an important basis for functional studies and retrofitting applications of EVs.
Reference：Luo T, Chen SY, Qiu ZX, et al. Transcriptomic Features in a Single Extracellular Vesicle via Single-Cell RNA Sequencing[J]. Small Methods. 2022 Sep 6:e2200881.
Case 3 Blocking connexin 43 and promoting ATP release from tubular epithelial cells improves renal fibrosis
It is unclear whether metabolites from injured tubular epithelial cells (TECs) are involved in renal fibrosis. After TEC injury, various metabolites are released, the most potent of which is adenosine triphosphate (ATP) released via ATP-permeable channels. Of these channels, connexin 43 (Cx43) is the most common member, however, its role in renal interstitial fibrosis (RIF) has not been fully investigated. The authors analysed kidney samples from patients with obstructive nephropathy and unilateral ureteral obstruction (UUO) mice and constructed a Cx43-KSP mouse model for the removal of Cx43 from TECs. the relationship between tubular Cx43, ATP and macrophages in renal interstitial fibrosis was explored by transcriptomic, metabolomic and single cell sequencing multi-omics analyses. The results showed that Cx43 expression was upregulated in both patients with obstructive nephropathy and mouse TECs, and knocking down Cx43 in TECs or using Cx43-specific inhibitors reduced UUO-induced inflammation and fibrosis in mice.
Single-cell sequencing (ScRNA-seq) results showed that ATP receptors were mainly concentrated in macrophages, and macrophages expressing ATP receptors were accompanied by upregulated expression of focal death genes. CXCL10 has been reported to be associated with organ fibres and can activate fibroblasts by binding to CXCR3 on fibroblasts. Cx43 mediates the release of ATP from TECs during renal injury, which in turn leads to the release of CXCL10, and activates intrarenal fibroblasts and accelerates renal fibrosis.
Reference：Xu H,Wang M,Li Y,et al.Blocking connexin 43 and its promotion of ATP release from renal tubular epithelial cells ameliorates renal fibrosis[J].Cell Death &Disease,2022.
Flow Chart of Single-cell Transcriptome Bioinformatics Analysis
